Mitochondria are subcellular organelles that generate ATP, control apoptotic cell death, buffer intracellular calcium and generate reactive oxygen species and free radicals. Perturbations in the function or regulation of these pathways have been implicated numerous diseases including cancer, diabetes, cardiac infarction, cerebral ischemia, Alzheimer's disease, amyotrophic lateral sclerosis and sepsis. The cytochromes of the electron transport chain are single electron carriers which have strong oxidation- dependent absorption bands at visible wavelengths. Two-wavelength spectrophotometry has been used extensively to study the cytochromes of the electron transport chain from isolated mitochondria and has yielded enormous amounts of information on mitochondrial function. However, this technology has not been extended to cells where the interaction between mitochondria and cytosolic proteins can be studied. Recently we have introduced a full-spectral technology with multi-wavelength fitting which has the sensitivity to measure oxidation changes in all the cytochromes of the electron transport chain from living cells. The goal of this application is to develop this technology to fruition so that it can be used study the interaction between cytosolic signaling pathways and mitochondrial function. We have three specific aims. (1) Develop instrumentation to oxygenate living cells and combine our spectroscopic measurement of cytochrome oxidation changes with a continuous measurement of cellular oxygen and glucose consumption so studies over the time course of the signaling cascades can be carried out. (2) Integrate our technology with fluorescence spectroscopy so that changes in mitochondrial function can mechanistically linked to cytosolic events measured with commercially available fluorescence based assays. (3) Develop and validate spectral analysis algorithms to calculate the absolute cytochrome oxidation state instead of our current measure of oxidation changes. PUBLIC HEALTH RELEVANCE: Mitochondria are cellular components which are essential for normal cell function and hence human health. Changes in mitochondrial function have been implicated in numerous diseases including cancer, diabetes, cardiac infarction, cerebral ischemia, Alzheimer's disease, amyotrophic lateral sclerosis and sepsis. Recently we have introduced instrumentation based on optical spectroscopy that can measure mitochondrial function in living cells with unprecedented precision. The goal of this application is to develop this technology to fruition so that it can be used study the regulation of mitochondria in normal cells and how this regulation fails in human disease.